Method of preparing an antigen-containing formulation

ABSTRACT

An antigen-containing formulation is provided, comprising: (a) an antigen; (b) a TH1-inducing adjuvant; and (c) a sparingly soluble amino acid or a derivative thereof. The adjuvant may be, for example, monophosphoryl lipid A, 3′-de-O-acetylated monophosphoryl lipid A, derivatives thereof, or any other adjuvant that enhances an individual&#39;s TH response to the antigen. Suitable amino acids include tyrosine, tryptophan, derivatives thereof, and the like. Methods for using the formulation are also provided; in a particularly preferred embodiment, the formulation is used as a vaccine.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119 to U.K.Patent Application No. 9820525.5, filed Sep. 21, 1998.

TECHNICAL FIELD

[0002] The present invention relates to a novel formulationparticularly, but not exclusively, for use in immunization.

BACKGROUND

[0003] The immune system has evolved specifically to detect andeliminate foreign or new material from a host. This material may be ofviral, bacterial, or parasitic origin and may reside outside or withinthe cells of the host, or be of neoplastic origin.

[0004] The immune response to antigen is generally either cell mediated(T-cell mediated killing) or humoral (antibody production viarecognition of whole antigen). The pattern of cytokine production by TH(T-Helper) cells involved in an immune response can influence which ofthese response types predominates: cell mediated immunity (TH1) ischaracterized by high IL-2 and IFNγ but low IL-4 production, whereas inhumoral immunity (TH2) the pattern is low IL-2 and IFNγ but high IL-4,IL-5, IL-10. Since the secretory pattern is modulated at the level ofthe secondary lymphoid organ or cells, pharmacological manipulation ofthe specific TH cytokine pattern can influence the type and extent ofthe immune response generated.

[0005] The TH1-TH2 balance refers to the interconversion of the twodifferent forms of helper T cells. The two forms have large scale andopposing effects on the immune system. If an immune response favors TH1cells, then these cells will drive a cellular response, whereas TH2cells will drive an antibody-dominated response. The type of antibodiesresponsible for some allergic reactions is induced by TH2 cells.

[0006] Vaccination is the best known and most successful application ofimmunological principles to human health. Naturally, to be introducedand approved, a vaccine must be effective and the efficacy of allvaccines is reviewed from time to time. Many factors affect vaccineefficacy. An effective vaccine must: induce the right sort of immunity;be stable on storage; and have sufficient immunogenicity. Withnon-living vaccines, in particular, it is often necessary to boost theirimmunogenicity with an adjuvant. This can also apply to some live, e.g.,attenuated, vaccines. An “adjuvant” is a substance that enhances theimmune response to an antigen.

[0007] During work in the 1920s on the production of animal antisera forhuman therapy, it was discovered that certain substances, notablyaluminum salts, added to or emulsified with an antigen, greatly enhanceantibody production, i.e., they act as adjuvants. Aluminum hydroxide isstill widely used with, for example, diphtheria and tetanus toxoids.

[0008] GB-A-1 377 074, corresponding to U.S. Pat. No. 3,792,159,describes a process for preparing coprecipitates of tyrosine having anallergen dispersed therein.

[0009] GB-A-1 492 973, corresponding to U.S. Pat. No. 4,070,455,describes a process for preparing coprecipitates of tyrosine having amodified allergen dispersed therein. The allergen is modified bytreatment with an agent, such as glutaraldehyde, which causesintra-molecular cross-linking and reduces the allergenicity of theproduct relative to the unmodified allergen.

[0010] 3 De-O-acylated monophosphoryl lipid A (3-DMPL) is known fromGB-A-2 220 211, corresponding to U.S. Pat. No. 4,912,094 and assigned toRibi Immunochem. Res. (“Ribi”). Chemically, 3-DMPL is a mixture of 3de-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains andis manufactured by Ribi Immunochem. Montana. A preferred form of 3de-O-acylated monophosphoryl lipid A is disclosed in InternationalPatent Publication No. WO 92/16556.

[0011] International Patent Publication No. WO 98/44947 describes aformulation for use in desensitization therapy of allergy sufferers thatcomprises an optionally modified allergen, tyrosine and 3 de-O-acylatedmonophosphoryl lipid A.

[0012] Considerable efforts have been made to produce better adjuvants,particularly for T-cell-mediated responses, but it should be stressedthat very few of these more recent adjuvants have been accepted forroutine human use.

[0013] It appears that the effect of adjuvants is due mainly to twoactivities: the concentration of antigen in a site where lymphocytes areexposed to it (the “depot” effect) and the induction of cytokines, whichregulate lymphocyte function. Newer antigen delivery systems such asliposomes and immune-stimulating complexes (ISCOMS) achieve the samepurpose by ensuring that antigens trapped in them are delivered toantigen-presenting cells. Bacterial products such as mycobacterial cellwalls, endotoxins, etc., are believed to act by stimulating theformation of cytokines. Cytokine induction may be particularly useful inimmunocompromised patients, who often fail to respond to normalvaccines. It is hoped that such cytokine induction might also be usefulin directing the immune response in the desired direction, e.g., indiseases where only TH1 or TH2 cell responsiveness is wanted (Roitt etal “Immunology,” 4th edition Wolfe Publishing, 1995).

[0014] We now provide a new antigen formulation that can tilt theTH1-TH2 balance in favor of a TH1 response. The formulation is useful inimmunotherapy, particularly the field of vaccines. It is also useful instudying immune responses and in the production of antibodies.

SUMMARY OF THE INVENTION

[0015] According to one aspect of the present invention there isprovided a composition comprising:

[0016] (a) an antigen;

[0017] (b) a TH1-inducing adjuvant; and

[0018] (c) a sparingly soluble amino acid or a derivative thereof.

[0019] Preferably, the antigen is derived from a bacterium or virus, orother pathogenic organism, or neoplasm or from knowledge of theirantigenic structures.

[0020] Preferably, the TH1-inducing adjuvant is monophosphoryl lipid A(MPL), 3′-de-O-acetylated monophosphoryl lipid A (3-DMPL), or aderivative or salt thereof.

[0021] Preferably, the sparingly soluble amino acid is tyrosine,tryptophan or a derivative thereof.

[0022] The present invention also provides a composition for use inmedicine. That is, the invention provides a method for using acomposition to treat or prevent or reduce susceptibility to a bacterialinfection, a viral infection or other disease such as cancer.

[0023] Preferably, the composition is in the form of a vaccine, and theinvention further provides a method for preparing an immunoglobulin,comprising immunizing an animal with a composition of the presentinvention.

[0024] The present invention also provides a method for preparing acomposition of the present invention comprising mixing a solution of anantigen and the TH1-inducing adjuvant with a solution of the sparinglysoluble amino acid or derivative in a strong aqueous acid whileneutralizing the mixture of solutions, thereby co-precipitating thesparingly soluble amino acid, antigen and adjuvant. This method mayfurther comprise adding a pharmaceutically acceptable carrier, diluentor excipient.

DETAILED DESCRIPTION OF THE INVENTION I. The Antigen-ContainingFormulation

[0025] The composition of the invention is an antigen-containingformulation comprised of an antigen, a TH-inducing adjuvant, and asparingly soluble amino acid or a derivative thereof. Theantigen-containing formulation is particularly useful as a vaccine;however, the formulation is useful in other contexts as well, e.g., intreating, preventing or reducing susceptibility to certain diseases anddisorders, including, but not limited to, bacterial infections, viralinfections, and cancer. The individual components of the formulationwill now be described by way of non-limiting example.

[0026] (A) The Antigen

[0027] Originally the term “antigen” was used for any molecule thatinduced B cells to produce a specific antibody. Now, however, the termmay be used to indicate any molecule that can be specifically recognizedby the adaptive elements of the immune response, i.e., by B cells or Tcells, or both. Thus, an “antigen” is a molecule that reacts withpreformed antibody at specific receptors on T and B cells. However, weexclude what are traditionally known as “allergens,” i.e., an agent,e.g., pollen dust, that causes IgE-mediated hypersensitivity.

[0028] An allergy is a response to an environmental antigen (allergen)due to pre-existing IgE antibody attached to mast cells. An immediatehypersensitivity reaction is produced by mast cell products (histamine,etc.) causing asthma, hay fever, serum sickness, systematic anaphylaxisor contact dermatitis. There are four types of such hypersensitivityreaction (Types I, II, III and IV). The first three areantibody-mediated; the fourth is mediated mainly by T cells andmacrophages. The present invention does not relate to the use of such“environmental antigens.”

[0029] Thus, preferably the “antigen” used in the present invention doesnot include an “allergen” derived from any allergy causing substance,such as pollen (e.g., ragweed or birch pollen), food, insect venom,mold, animal fur or house dust mite (D. farinae or D. pteronyssinus).

[0030] The present invention can therefore be seen as relating toantigens that often involve a cellular, IgG2a or IgG2b mediatedresponse, rather than an IgE or IgG1 mediated response.

[0031] The antigen used in the present invention is preferably animmunogen, i.e., an antigen that activates immune cells to generate animmune response against itself.

[0032] In a preferred embodiment, the present invention relates to aformulation for use as a vaccine and the antigen is one that is usefulin such a vaccine.

[0033] The antigen used in the present invention can be any appropriateantigen, which is or becomes available.

[0034] The type of antigen used in a vaccine depends on many factors. Ingeneral, the more antigens of the microbe retained in the vaccine thebetter, and living organisms tend to be more effective than killed ones.Exceptions to this rule are diseases where a toxin is responsible forany pathogenic effect. In this case the vaccine can be based on thetoxin or toxoid alone.

[0035] The antigen used in the present invention may be derived from anyliving organisms; intact or non-living organisms; subcellular fragments;toxoids; recombinant DNA-based antigens or anti-idiotypes or syntheticantigens. The antigen may be derived from natural or attenuatedorganisms, which may be viral or bacterial. The type of antigen may be acapsular polysaccharide, surface or internal antigen. If recombinantDNA-based, the antigen may be obtained from a cloned and expressed geneor naked DNA.

[0036] The antigen may be modified by reaction, for example, with across-linking agent, such as a dialdehyde, more particularlyglutaraldehyde.

[0037] For example, micro-organisms against which vaccines are availableor are sought include Salmonella, Shigella, Klebsiella, Enterobacter,Serratia, Proteus, Yersinia, Vibrio, Aeromonas, Pasteurella,Pseudomonas, Acinetobacter, Moraxella, Flavobacterium, Bordetella,Actinobacillus, Neisseria, Brucella, Haemophilus and Escherichia coli.

[0038] Preferred vaccines include, but are not limited to: vaccinia (forsmallpox); vole bacillus (for tuberculosis); polio; measles, mumps;rubella; yellow fever; varicella-zoster; BCG (Bacillus Calmette-Gruérin,an antituberculosis vaccine); rabies; influenza; hepatitis A; typhus;pertussis; typhoid; cholera; plague; pneumococcus; meningococcus;Haemophilus influensae; hepatitis B; hepatitis C; tetanus anddiphtheria. Toxin-based vaccines include Chlostridium tetani,Corynebacterium diphtheriae, Vibrio cholerae and Clostridiumperfringens.

[0039] Other major diseases for which the vaccines of the invention maybe useful include, but are not limited to: HIV, herpes, viruses,adenoviruses, rhinoviruses, staphylococci, group A streptococci,Mycobacterium leprae, Treponema pallidum, Chlamydia, Candida,Pneumocystis, malaria, trypanosomiasis; Chagas' disease; schistosomiasisand onchoceriasis.

[0040] The presence of tumor antigens also has been demonstrated, and,as a result, the concept of vaccinating against cancer has arisen. Also,in principle, conception and implantation can be interrupted by inducingimmunity against a wide range of pregnancy and other reproductivehormones.

[0041] (B) The Th1-Inducing Adjuvant

[0042] By “TH1-inducing adjuvant” is meant an adjuvant that enhances theTH1 response to an antigen.

[0043] The effectiveness of an adjuvant as a TH1-inducing adjuvant maybe evaluated by determining the profile of antibodies directed againstan antigen resulting from administration of this antigen in vaccinesthat are also comprised of the various adjuvants.

[0044] Preferably the adjuvant is a modified lipopolysaccharide. Asdescribed in U.S. Pat. No. 4,912,094, enterobacterial lipopolysaccharide(LPS) is a powerful immunostimulant. However, it can also elicit harmfuland sometimes fatal responses. It is now known that the endotoxicactivities associated with LPS result from its lipid A component.Accordingly, the present invention more preferably uses a detoxifiedderivative of lipid A. Ribi produced a derivative of lipid A originallyknown as refined detoxified endotoxin (RDE) but which has become knownas monophosphoryl lipid A (MPL). As described in U.S. Pat. No.4,912,094, MPL is produced by refluxing LPS or lipid A obtained fromheptoseless mutants of gram negative bacteria (e.g., Salmonella sp.) inmineral acid solutions of moderate strength (e.g., 0.1N HCl) for aperiod of around 30 minutes. This treatment results in loss of thephosphate moiety at position 1 of the reducing-end glucosamine. Inaddition, the core carbohydrate is removed from the 6′ position of thenon-reducing glucosamine during this treatment.

[0045] Preferably, however, a modified LPS or lipid A is used in whichthe detoxified lipid A retains the core moiety attached to the 6′position of non-reducing glucosamine. Such derivatives of LPS and lipidA are also described in U.S. Pat. No. 4,912,094. In more detail, U.S.Pat. No. 4,912,094 discloses a modified lipopolysaccharide that isobtained by selectively removing only the β-hydroxymyristic acyl residueof lipopolysaccharide that is ester-linked to the reducing-endglucosamine at position 3′ of said lipopolysaccharide, which comprisessubjecting said lipopolysaccharide to alkaline hydrolysis. Suchde-O-acylated monophosphoryl lipid A, diphosphoryl lipid A (DPL) and LPSmay be used as the TH1-inducing adjuvant in the present invention. Thusin a preferred embodiment, the present invention uses MPL, DPL or LPS inwhich the position 3′ of the reducing end glucosamine is de-O-acylated.These compounds are known as 3-DMPL, 3-DDPL and 3-DLPS respectively.

[0046] In U.S. Pat. No. 4,987,237 derivatives of MPL having the formula

[0047] are described, wherein R¹ and R² are H or lower alkyl, and R³ isstraight or branched chain hydrocarbon composed of C, H and optionallyO, N and S, which if more than one atom may be the same or different,wherein the total number of carbon atoms does not exceed 60, and thecircle represents an MPL nucleus.

[0048] Alternatively, the MPL derivative has the formula

[0049] wherein the segment of the derivative represented by

[0050] contains 2-60 carbon atoms and wherein R³ is straight or branchedchain hydrocarbon composed of C, H and optionally O, N and S, which ifmore than one atom may be the same or different, and x is a minimum of 1and can be any whole number such that the total number of C atoms in allx segments does not exceed 60, and wherein the chemical structure ofeach R³ may be the same or different in each such segment and whereinthe circle represents an MPL nucleus.

[0051] All such derivatives of LPS or lipid A which are or becomeavailable may be used as the TH1-inducing adjuvants in the presentinvention.

[0052] The TH1-inducing adjuvant can be mixed with the other componentsof the composition prior to administration. Alternatively, it can beformulated together with the other components during manufacture of theproduct. Alternatively, it can be administered at a different site ortime than the other components. Administration can be by a number ofroutes.

[0053] (C) The Sparingly Soluble Amino Acid

[0054] The amino acid of the present formulation must be sparinglysoluble in aqueous solution such that the adjuvant and antigen areallowed to produce an immune response.

[0055] Most of the amino acids are only sparingly soluble in water, as aconsequence of the strong intermolecular forces acting in the crystallattice. Exceptions are glycine, proline, lysine, threonine, cysteineand arginine, which are all quite soluble in water, and do not form partof the invention. The water solubility of amino acids is given in thefollowing Table: Amino Acid Water Solubility g/100 ml H₂O at 25° C.glycine 25 alanine 16.7 valine 8.9 leucine 2.4 isoleucine 4.1 methionine3.4 proline 162 phenylalanine 3.0 tryptophan 1.1 serine 5.0 threoninevery cysteine very tyrosine 0.04 asparagine 3.5 glutamine 3.7 asparticacid 0.54 glutamic acid 0.86 lysine very arginine 15 histidine 4.2

[0056] Preferably the water solubility of the amino acid used in theinvention is about 1.1 or less g/100 ml H₂O at 25° C. Particularlypreferred are tyrosine or tryptophan; the more insoluble tyrosine beingpreferred. Derivatives of these amino acids such asbenzyl-O-octadecanoyl-L-tyrosine are also included within the scope ofthe present invention.

[0057] Typically, the antigen is dispersed within and/or adsorbed ontothe amino acid, e.g., by co-precipitation or mixing, respectively.

II. Preparation

[0058] The composition of the present invention may be prepared bymixing an aqueous solution of the antigen with a solution of the aminoacid in a strong aqueous acid, neutralizing the mixture of solution,thereby co-precipitating the amino acid and antigen, mixing the productwith the TH1-inducing adjuvant, and optionally adding a physiologicallyacceptable diluent, excipient or carrier, before or after theaforementioned mixture. Alternatively, the TH1-inducing adjuvant may beco-precipitated with the antigen. As well as being mixed orco-precipitated with the other components of the composition prior toadministration, the TH1-inducing adjuvant can be administered at adifferent site and/or time to the other components.

[0059] Typically an aqueous solution of the antigen, preferably at pH7±1, obtainable from the solvation of a solid, is mixed with a solutionof the amino acid in a strong aqueous acid. The strong acid is usuallyan inorganic acid, preferably hydrochloric acid. The solution of antigenused in this step typically contains between 0.1 μg/ml and 1000 μg/mlantigen protein, for example about 400 μg/ml. The ratio of antigen:aminoacid in the mixture is typically in the range of approximately 1:4×10⁵to 1:1×10² w/w.

[0060] The resulting mixture of solutions of antigen and amino acid isneutralized. By neutralization is meant an adjustment of pH to a valuewithin the range 4.0 to 7.5. It is desirable that, at no time, or atleast no prolonged time, during the neutralization does the pH of thesolution rise appreciably above 7.5. This condition can be met byvigorous stirring of the solution and by the use of only the requiredamount of base, if desired. Various buffering agents can usefully beadded to the solutions of antigen to assist in pH control during mixingand neutralizing stages.

[0061] A particularly useful method of carrying out the neutralizationis for separate streams of the solution of amino acid and neutralizingbase to be run into the solution of antigen. The rates of flow of theadded solutions are controlled by pH-state, that is, by equipment thatregulates the flow of one or both of the solutions so that the pH of thereaction mixture remains substantially constant at a predeterminedlevel. Optimum results are generally obtained by maintaining the pHwithin the range of approximately 6.5 to 7.5, although the precise pHmay vary according to the nature of the antigen.

[0062] The result of neutralization is the immediate precipitation ofthe amino acid, within and/or upon which the solution of antigen isoccluded and/or adsorbed. After precipitation, the mixture is eitherwashed immediately or allowed to stand for a period of from a few hoursto a day or two prior to washing.

[0063] The resulting precipitate may be removed from the solution bycentrifugation or filtration and washed, e.g., with phenol-saline,before resuspending, if required, in a physiologically acceptablecarrier, excipient or diluent.

[0064] MPL (or other TH1-inducing adjuvant) that has been dissolved bythe method described in Preparation 3 below or by sonification can bediluted by various means prior to its addition to amino acid adsorbatesof antigens. The preparation of MPL is initially made at a concentrationof typically between 0.5 mg per ml and 4 mg per ml, for example 1 mg perml. It can then be diluted to a concentration of between 500 μg/ml and20 μg/ml, preferably 100 μg/ml. This dilution can be made in pure water,or in an aqueous glycerol solution containing between 1% and 4%,preferably 2%, glycerol. Such dilutions can then be added to asuspension of the amino acid adsorbate prepared as described above. Forconvenience, the concentration of the MPL solution and the amino acidadsorbate suspension respectively may be selected such thatapproximately equal volumes of each of admixed to obtain the finalproduct for injection. A typical final product contains about 100 μg/mlof antigen and about 250 μg/ml of MPL.

[0065] Thus, although the formulation of the invention may beadministered directly, preferably the formulation is combined with apharmaceutically acceptable carrier, excipient or diluent to produce apharmaceutical composition, which may be for human or veterinary use.Suitable physiologically acceptable carriers and diluents includeisotonic saline solutions, for example phosphate-buffered saline,phenol-saline and sterile water. The compositions may be formulated forparenteral, intramuscular, intravenous, subcutaneous, intraocular ortransdermal administration.

[0066] The routes of administration and dosages described herein areintended only as a guide since a skilled practitioner will readily beable to determine the optimum route of administration and dosage for anyparticular patient and condition.

III. Vaccines

[0067] Vaccines may be prepared from the formulation of the presentinvention. The preparation of vaccines that contain an antigen as activeingredient is known to one skilled in the art. Typically, such vaccinesare prepared as injectables, either as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid prior toinjection may also be prepared. The preparation may also be emulsified,or the formulation encapsulated in liposomes. As indicated above, theformulation may be mixed with carriers, diluents and excipients that arepharmaceutically acceptable and compatible with the formulation. Suchexcipients include, for example, water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof.

[0068] In addition, if desired, the vaccine may contain minor amounts ofauxiliary substances such as wetting or emulsifying agents, pH bufferingagents and/or further adjuvants that enhance the effectiveness of thevaccine.

[0069] The proportion of antigen and adjuvant can be varied over a broadrange so long as both are present in effective amounts. Conveniently,the vaccines are formulated to contain a final concentration of antigenin the range of 0.2 μg/ml to 200 μg/ml, preferably 5 μg/ml to 50 μg/ml,most preferably about 15 μg/ml.

[0070] After formulation, the vaccine may be incorporated into a sterilecontainer that is then sealed and stored at low temperature, for example40° C., or it may be freeze-dried. Lyophilization permits long-termstorage in a stabilized form.

[0071] The vaccines are conventionally administered parenterally, byinjection, for example, either subcutaneously or intramuscularly.Additional formulations that are suitable for other modes ofadministration include suppositories and, in some cases, oralformulations. For suppositories, traditional binders and carriers mayinclude, for example, polyalkylene glycols or triglycerides; suchsuppositories may be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1% to 2%. Oralformulations include such normally employed excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain 10% to95% of active ingredient, preferably 25% to 70%. Where the vaccinecomposition is lyophilized, the lyophilized material may bereconstituted prior to administration, e.g., as a suspension.Reconstitution is preferably effected in buffer.

[0072] Capsules, tablets and pills for oral administration to a patientmay be provided with an enteric coating comprising, for example,copolymers of methacrylic acid and methyl methacrylate available underthe trademark Eudragit S, and Eudragit L, cellulose acetate, celluloseacetate phthalate or hydroxypropylmethyl cellulose.

[0073] The antigens used in the invention may be formulated into thevaccine as neutral or salt forms. Pharmaceutically acceptable saltsinclude the acid addition salts (formed with free amino groups of thepeptide) and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or organic acids such as acetic,oxalic, tartaric and maleic. Salts formed with the free carboxyl groupsmay also be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidineand procaine.

IV. Dosage and Administration of Vaccines

[0074] The vaccines are administered in a manner compatible with thedosage formulation, and in such amount as will be prophylacticallyand/or therapeutically effective. The quantity to be administered, whichis generally in the range of 5 μg to 250 μg of antigen per dose, dependson the subject to be treated, capacity of the subject's immune system tosynthesize antibodies, and the degree of protection desired. Apreferable range is from about 20 μg to about 40 μg per dose.

[0075] A suitable dose size is about 0.5 ml. Accordingly, a dose forintramuscular injection, for example, would comprise 0.5 ml containing20 μg of immunogen in admixture with 0.5% adjuvant.

[0076] Precise amounts of active ingredient required to be administeredwill depend on the judgment of the practitioner and may vary with eachsubject.

[0077] The vaccine may be given in a single dose schedule, or preferablyin a multiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals required to maintainand or reinforce the immune response, for example, at 1 to 4 months fora second dose, and if needed, a subsequent dose(s) after several months.The dosage regimen will also, at least in part, be determined by theneed of the individual and be dependent upon the judgement of thepractitioner.

[0078] In addition, the vaccine containing the antigen(s) may beadministered in conjunction with other immunoregulatory agents, forexample, immunoglobulins.

V. Preparation of Antibodies Using the Formulation of the Invention

[0079] Compositions according to the invention may be used directly asimmunogens, without the use of further adjuvants to generate antiseraand monoclonal antibodies. The invention thus provides a method forinducing antigen specific immunoglobulin production comprising the stepsof:

[0080] a) immunizing an animal with a composition according to thepresent invention; and

[0081] b) recovering immunoglobulin specific for a region of the antigenof the composition from the serum of the animal.

[0082] The animals used for antibody production may be any animalsnormally employed for the purpose, particularly mammals. Especiallyindicated are mice, rats, guinea pigs and rabbits.

[0083] Immunization is carried out according to established techniques(See “Antibodies, A Laboratory Manual” by E. Harlow and D. Lane (1988)Cold Spring Harbor, U.S.A.). The purified composition (about 1 mg) wasinjected into a rabbit. A booster injection of 0.5 mg of the compositionwas made 4 weeks after the initial injection. Antibodies are isolatedfrom rabbit serum and tested for reactivity. Antibodies capable ofselective binding to the chosen antigen are obtained by this method.

[0084] More particularly, the formulation of the present inventioncomprising the antigen can be used to produce antibodies, bothpolyclonal and monoclonal. If polyclonal antibodies are desired, aselected mammal (e.g., mouse, rabbit, goat, horse, etc.) is immunized.Serum from the immunized animal is collected and treated according toknown procedures. If serum containing polyclonal antibodies to otherantigens, the polyclonal antibodies can be purified by immunoaffinitychromatography. Techniques for producing and processing polyclonalantisera are known in the art.

[0085] Monoclonal antibodies directed against antigens used in theinvention can also be readily produced by one skilled in the art. Thegeneral methodology for making monoclonal antibodies by hybridomas iswell known. Immortal antibody-producing cell lines can be created bycell fusion, and also by other techniques such as direct transformationof B lymphocytes with oncogenic DNA, or transfection with Epstein-Barrvirus. Panels of monoclonal antibodies produced against antigens can bescreened for various properties; i.e., for isotype and epitope affinity.

[0086] An alternative technique involves screening phage displaylibraries where, for example, the phage express scFv fragments on thesurface of their coat with a large variety of complementary determiningregions (CDRs). This technique is well known in the art.

[0087] Antibodies, both monoclonal and polyclonal, which are directedagainst antigens are particularly useful in diagnosis, and those whichare neutralizing are useful in passive immunotherapy. Monoclonalantibodies, in particular, may be used to raise anti-idiotypeantibodies. Anti-idiotype antibodies are immunoglobulins that carry an“internal image” of the antigen of the infectious agent against whichprotection is desired.

[0088] Techniques for raising anti-idiotype antibodies are known in theart. These anti-idiotype antibodies may also be useful for treatment, aswell as for an elucidation of the immunogenic regions of antigens.

[0089] For the purposes of this invention, the term “antibody,” unlessspecified to the contrary, includes fragments of whole antibodies thatretain their binding activity for a target antigen. Such fragmentsinclude Fv, F(ab′) and F(ab′)2 fragments, as well as single chainantibodies (scFv). Furthermore, the antibodies and fragments thereof maybe humanized antibodies, for example as described in EP-A-239400.

[0090] It is to be understood that while the invention has beendescribed in conjunction with the preferred specific embodimentsthereof, that the description above as well as the examples which followare intended to illustrate and not limit the scope of the invention.Other aspects, advantages and modifications within the scope of theinvention will be apparent to those skilled in the art to which theinvention pertains.

[0091] All patents, patent applications, and publications mentionedherein, both supra and infra, are hereby incorporated by reference.

REFERENCE EXAMPLE

[0092] Preparation 1

[0093] Eight mg of ovalbumin (XOA) as a model allergen were dissolved bymixing in 20 ml of EVANS solution, a standard physiological bufferedsaline solution. Next, 6.9 ml of phosphate buffer was added with mixing.The solution was placed in a 100 ml beaker containing a magnetic stirbar. While mixing using a magnetic stirrer, 6.9 ml or 3.2N NaOH and 6.9ml of 3.8N HCl, containing 24% w/v tyrosine were added simultaneously,dropwise, over a period of 5 minutes to form a precipitate. The mixturewas allowed to stir for an additional 5 minutes and then transferred toa 50 ml centrifuge tube and centrifuged for 10 minutes at 2500 rpm.After centrifugation the supernatant was decanted and the pelletedprecipitate resuspended in 40 ml of phosphate buffer. The mixture wascentrifuged for 5 minutes at 2500 rpm. After centrifugation thesupernatant was decanted and the precipitate resuspended in 40 ml ofphosphate buffer. The mixture was centrifuged for 5 minutes at 2500 rpm.After centrifugation the supernatant was decanted and the pelletedprecipitate resuspended in 40 ml of phosphate buffer saline, pH 7.2containing 0.4% v/v glycerol and 0.01% w/v thimerosal as a preservative.The final product contained approximately 40 mg/ml of tyrosineadsorbate. Assuming 100% binding of the XOA to the tyrosine adsorbatethe XOA was at 200 μg/ml in the final product. The XOA tyrosineadsorbate was stored at 4° C. until needed.

[0094] Preparation 2

[0095] A 4 mg/ml solution of 1.2-dipalmitoyl-SN-glycero-3-phosphocholine (DPPC) in absolute ethanol was prepared. For each 1.0 mg ofMPL®-TEA (triethylamine) salt to be solubilized, 27 μl of DPPC wereadded to dissolve the MPL®. The ethanol was removed by blowing a streamof N₂ gently into the vial. Next 1.0 ml of pyrogen-free water forinjection was added for each mg of MPL® in the dried MPL®/DPPC mixture.The solution was sonicated in a bath sonicator at 60-70° C. until clear.The MPL®/DPPC solution was then filter sterilized by filtration througha SFCA 290-4520 Nalgene 0.2 μm filter. The MPL®/DPPC solution wasaseptically dispensed at 1.0 mg/ml into depyrogenated vials, labelledMPL®-AF, and stored at 4° C.

[0096] Biological Activity

[0097] TH1 inducing activity in mice can be equated with the productionof IgG2a and IgG2b antibodies and the TH2 inducing activity with theproduction of IgG1 antibodies and IgE antibodies.

[0098] Therefore, as an example, an experiment was carried out in miceto demonstrate the profiles of the allergen specific antibodies to anexemplar ovalbumin (XOA) which is a well-known allergen derived fromchicken eggs. It was confirmed that a formulation consisting ofMPL+XOA+tyrosine stimulated a more advantageous antibody profile thanMPL+XOA, XOA+tyrosine or XOA alone.

[0099] Groups of 8 BALB/c female mice, 6-8 weeks of age, were injectedsubcutaneously in the inguinal area with 0.2 ml of one of the followingvaccines.

[0100] XOA+Tyrosine

[0101] The XOA tyrosine adsorbate prepared in Preparation 1 above wasdiluted with an equal volume of phosphate buffered saline within 30minutes prior to injection.

[0102] XOA+Tyrosine+MPL

[0103] The XOA tyrosine adsorbate prepared in Preparation 1 above wasdiluted with an equal volume of MPL®-AF at 500 μg/ml in phosphatebuffered saline within 30 minutes prior to injection.

[0104] XOA+MPL

[0105] XOA was dissolved in phosphate buffered saline at 200 μg/ml anddiluted with an equal volume of MPL®-AF at 500 μg/ml in phosphatebuffered saline within 30 minutes prior to injection.

[0106] XOA Alone

[0107] XOA was dissolved at 200 μg/ml in phosphate buffered saline anddiluted with an equal volume of phosphate buffered saline.

[0108] Twenty-one days later the four groups of mice were boosted with0.2 ml of freshly prepared vaccines. Fourteen days following the boosterthe mice were bled and the sera separated and stored at −70° C. untilassay.

[0109] The sera were assayed by conventional ELISA technique usinghorseradish conjugated goat anti-mouse IgG₁, IgG_(2a) and IgG_(2b)antibodies purchased from Southern Biotechnology Inc. (Birmingham, Ala.)and used according to the manufacturer's instruction. The IgG1, IgG2aand IgG2b titers represent the reciprocal serum dilution giving areading of >0.1 OD units at A₄₉₀.

[0110] The serum IgE levels were measured using an anti-IgE captureELISA followed by the use of a biotinylated ovalbumin probe. Binding wasmeasured following the addition of a horseradish conjugated strepavidinpreparation. The results are reported as OD units at A₄₉₀.

[0111] Results

[0112] Of particular importance is the fact that the combination ofallergen+tyrosine+MPL induces less antigen specific IgE antibody thanthe other combinations. Furthermore, the ratio of IgG2a or IgG2b to IgG1antibodies is greater and consistent with the highest levels of the twoformer antibody isotypes seen in the experiment in the mice givenantigen+tyrosine+MPL than in any other group of mice. This is indicativeof a better ratio of TH1 cell induction over TH2 cell induction in thisgroup compared with that induced in the other groups of mice.

EXAMPLES ACCORDING TO THE PRESENT INVENTION

[0113] Preparation A

[0114] To a neutral solution of purified polypeptide that displayshepatitis B virus antigenicity (details of how to prepare such apolypeptide can be found in EP-A-0 182 442 and WO 98/44947) is addedphosphate buffer solution at a pH of 7±1. The antigen solution isco-precipitated with tyrosine by the simultaneous addition of one volumeof 1-tyrosine in HCl (prepared by dissolving 24 g L-tyrosine to 100 mlwith 3.8M HCl) and one volume of 3.2M NaOH, to four volumes of antigensolution, with vigorous agitation. The suspension so formed iscentrifuged and then washed repeatedly with buffered saline pH 6±1.

[0115] Preparation B

[0116] As for Preparation 1 of Reference Example, except that XOA isreplaced with a polypeptide that displays hepatitis virus (HBV)antigenicity.

[0117] Preparation C

[0118] Same as Preparation 2 of Reference Example.

[0119] Biological Activity

[0120] TH1 inducing activity in mice can be equated with the productionof IgG2a and IgG2b antibodies and the TH2 inducing activity with theproduction of IgG1 antibodies and IgE antibodies.

[0121] Therefore, as an example, an experiment is carried out in mice todemonstrate the profiles of the antigen specific antibodies to anexemplar antigen (HBV) which is a well-known antigen. It is confirmedthat a formulation consisting of MPL+HBV+tyrosine stimulates a moreadvantageous antibody profile than MPL+HBV, HBV+tyrosine or HBV alone.

[0122] Groups of 8 BALB/c female mice, 6-8 weeks of age, are injectedsubcutaneously in the inguinal area with 0.2 ml of one of the followingvaccines.

[0123] HBV+Tyrosine

[0124] The HBV tyrosine adsorbate prepared in Preparation A above isdiluted with an equal volume of phosphate buffered saline within 30minutes prior to injection.

[0125] HBV+Tyrosine+MPL

[0126] The HBV tyrosine adsorbate prepared in Preparation A above isdiluted with an equal volume of MPL®-AF at 500 μg/ml in phosphatebuffered saline within 30 minutes prior to injection.

[0127] HBV+MPL

[0128] HBV is dissolved in phosphate buffered saline at 200 μg/ml anddiluted with an equal volume of MPL®-AF at 500 μg/ml in phosphatebuffered saline within 30 minutes prior to injection.

[0129] HBV Alone

[0130] HBV is dissolved at 200 μg/ml in phosphate buffered saline anddiluted with an equal volume of phosphate buffered saline.

[0131] Twenty one days later the four groups of mice are boosted with0.2 ml of freshly prepared vaccines. Fourteen days following the boosterthe mice are bled and the sera separated and stored at −70° C. untilassay. The sera are assayed by conventional ELISA technique usinghorseradish conjugated goat anti-mouse IgG₁, IgG₂a and IgG₂b antibodiespurchased from Southern Biotechnology Inc. (Birmingham, Ala., U.S.A.)and used according to the manufacturer's instructions. The IgG₁,IgG_(2a) and IgG_(2b) titers represent the reciprocal serum dilutiongiving a reading of >0.1 OD units at A₄₉₀. The serum IgE levels aremeasured using an anti-IgE capture ELISA followed by the use of abiotinylated ovalbumin probe. Binding is measured following the additionof a horseradish conjugated strepavidin preparation. Of particularimportance is the fact that the combination of antigen+tyrosine+MPL mayinduce less antigen specific IgE antibody than the other combinations.Furthermore, the ratio of IgG2a or IgG2b to IgG1 antibodies may begreater and consistent with the highest levels of the two formerantibody isotypes seen in the experiment in the mice givenantigen+tyrosine+MPL than in any other group of mice. This is indicativeof a better ratio of TH1 cell introduction over TH2 cell induction inthis group compared with that induced in other groups of mice.

1. A composition comprising: (a) an antigen; (b) a TH1-inducingadjuvant; and (c) a sparingly water soluble amino acid or a derivativethereof.
 2. The composition of claim 1, wherein the antigen is derivedfrom a bacterium, virus or neoplasm.
 3. The composition of claim 1,wherein the antigen comprises a polypeptide.
 4. The composition of claim2, wherein the antigen comprises a polypeptide.
 5. The composition ofclaim 1, wherein the antigen is in the form of a vector comprising apolynucleotide encoding an antigenic polypeptide and operably linked toa regulatory sequence permitting expression of the polynucleotide. 6.The composition of claim 2, wherein the antigen is in the form of avector comprising a polynucleotide encoding an antigenic polypeptide andoperably linked to a regulatory sequence permitting expression of thepolynucleotide.
 7. The composition of any one of claims 1, 2, 3, 4, 5 or6, wherein the TH1-inducing adjuvant is MPL, 3-DMPL or a derivative orsalt thereof.
 8. The composition of any one of claims 1, 2, 3, 4, 5 or6, wherein the sparingly soluble amino acid is tyrosine, tryptophan or aderivative thereof.
 9. The composition of claim 7, wherein the sparinglysoluble amino acid is tyrosine, tryptophan or a derivative thereof. 10.The composition of claim 1, further comprising a pharmaceuticallyacceptable carrier, diluent or excipient.
 11. The composition of claim10, comprising a vaccine.
 12. A method for treating or preventing orreducing the susceptibility to bacterial or viral infection or cancer ina human or animal which comprises administering to the human or animalan effective amount of a composition comprising an antigen, aTH1-inducing adjuvant, and a sparingly water soluble amino acid or aderivative thereof.
 13. The method of claim 12, wherein the antigen isderived from a bacterium, virus or neoplasm.
 14. The method of claim 12,wherein the antigen comprises a polypeptide.
 15. The method of claim 13,wherein the antigen comprises a polypeptide.
 16. The method of claim 12,wherein the antigen is in the form of a vector comprising apolynucleotide encoding an antigenic polypeptide and operably linked toa regulatory sequence permitting expression of the polynucleotide. 17.The method of claim 13, wherein the antigen is in the form of a vectorcomprising a polynucleotide encoding an antigenic polypeptide andoperably linked to a regulatory sequence permitting expression of thepolynucleotide.
 18. The method of any one of claims 12, 13, 14, 15, 16or 17, wherein the TH1-inducing adjuvant is MPL, 3-DMPL or a derivativethereof.
 19. The method of any one of claims 12, 13, 14, 15, 16 or 17,wherein the sparingly soluble amino acid is tyrosine, tryptophan or aderivative thereof.
 20. The method of claim 18, wherein the sparinglysoluble amino acid is tyrosine, tryptophan or a derivative thereof. 21.The method of claim 12, wherein the composition further comprises apharmaceutically acceptable carrier, diluent or excipient.
 22. A methodfor producing antibodies in a mammalian individual, comprisingadministering to the individual an effective amount of a compositioncomprising an antigen, a TH1-inducing adjuvant, and a sparingly watersoluble amino acid or a derivative thereof, wherein the antibodiesproduced recognize the antigen.
 23. A method of treating bacterial orviral infection or cancer in a human or animal which comprisesadministering to a human or animal an effective amount of an antibodyproduced according to the method of claim
 22. 24. A method for preparingthe composition of claim 1, comprising mixing a solution of an antigenand the TH1-inducing adjuvant with a solution of the sparingly solubleamino acid or derivative thereof in a strong aqueous acid whileneutralizing the mixture of solutions, thereby co-precipitating thesparingly soluble amino acid, antigen and adjuvant.
 25. The method ofclaim 24, further comprising adding a pharmaceutically acceptablecarrier, diluent or excipient to the mixture.